The invention relates to the purification of alpha-1 proteinase inhibitor (xcex1-1 PI) from aqueous solutions. More specifically, the invention relates to the purification of xcex1-1 PI from blood plasma or from plasma fractions produced from Cohn-Oncley fractionation with chromatography. Viral removal is accomplished by the addition of PEG and by nanofiltration. Inactivation of enveloped viruses is accomplished by addition of detergent prior to the chromatography process.
Alpha-1 proteinase inhibitor (xcex1-1 PI) is a glycoprotein with a molecular weight of about 55,000 Daltons. The protein is a single polypeptide chain to which several oligosaccharide units are covalently bound. Alpha-1 PI acts as an inhibitor of endogenous proteases, such as trypsin, chymotrypsin, pancreatic elastase, skin collagenase, renin, urokinase and proteases of polymorphonuclear lymphocytes.
Alpha-1 PI is currently used therapeutically to treat persons having a genetically caused deficiency of xcex1-1 PI. In such a condition, xcex1-1 PI is administered to inhibit lymphocyte elastase in the lungs. Lymphocyte elastase breaks down foreign proteins in the lungs. When xcex1-1 PI is not present in sufficient quantities to regulate elastase activity, the elastase breaks down lung tissue. In time, this imbalance results in chronic lung tissue damage and emphysema. Alpha-1 PI has been successfully used to treat this form of emphysema.
The demand for xcex1-1 PI typically exceeds the available supply. Alpha-1 PI for therapeutic use is currently purified from human plasma. This source of the protein is limited, which contributes to the low supply. In order to maximize the available supply of xcex1-1 PI, a process for purifying xcex1-1 PI from human plasma should have the highest possible yield. The purity of the xcex1-1 PI isolated from human plasma is also critical, because trace impurities can stimulate immune responses in patients who are receiving xcex1-1 PI. Finally, the process of purifying xcex1-1 PI from human plasma using current techniques requires an extensive amount of time, for the separation of the xcex1-1 PI from other proteins, viruses, etc. All of these factors (i.e., low yields, long production times, and low purity), contribute to the inadequate supply of xcex1-1 PI.
Various methods of purifying xcex1-1 PI from human plasma have been described. Bollen, et al., U.S. Pat. No. 4,629,567 (1986) used five different chromatography steps to purify the xcex1-1 PI from yeast, E. coli, and human plasma. The five steps involved DEAE ion exchange, thiol-disulfide exchange, heparin affinity, zinc-chelate chromatography, and amino hexyl ion exchange. No purity and yield data were shown.
Novika, et al., Gematol. Transfuziol. 34:46-50 (1989) reported isolation methods from the by-products of the manufacture of blood products. They used affinity, DEAE cellulose, and gel filtration chromatographies. The purity and yield data were not available.
Podiarene, et al., Vopr. Med. Khim. 35:96-99 (1989) reported a single step procedure for isolation of xcex1-1 PI from human plasma using affinity chromatography with monoclonal antibodies. Alpha-1 PI activity was increased 61.1 fold with a yield of 20%.
Burnouf, et al., Vox. Sang. 52, 291-297 (1987) starting with plasma supernatant A (equivalent to Cohn Fraction II+III) used DEAE chromatography and size exclusion chromatography to produce an xcex1-1 PI which was 80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recovery was 65-70% from the supernatant A.
Hein, et al., Eur. Respir. J. 9:16s-20s (1990) and co-owned U.S. Pat. No. 4,697,003 present a process which employs Cohn Fraction IV-1 as the starting material and utilizes fractional precipitation of xcex1-1 PI with polyethylene glycol followed by anion exchange chromatography on DEAE Sepharose(copyright). The final product has a purity of about 60% with 45% yield.
Dubin, et al., Prep. Biochem. 20:63-70 (1990) have shown a two step chromatographic purification. First xcex1-1 PI, CI inhibitor, xcex1-1 antichymotrypsin, and inter xcex1-1 trypsin inhibitor were eluted from Blue Sepharose(copyright) and then xcex1-1 PI was purified by gel filtration. Purity and yield data were not available.
Ballieux, et al., purified an xcex1-1 PI and proteinase-3 complex from purulent sputum using 4-phenylbutylamine affinity chromatography, cation exchange, and a final immunoaffinity step (Ballieux, B. E., et al., J. Immunol. Methods 159:63-70 (1993)). The pH of the buffer used in the cation exchange step was 7.0. Under the conditions used, most of the sputum proteins bound to the resin, but xcex1-1 PI and proteinase-3 passed through without binding.
Jordan, et al., U.S. Pat. No. 4,749,783 (1988) described a method where biologically inactive proteins in a preparation were removed by affinity chromatography after a viral inactivation step. The basis of the separation between the native and denatured forms of the protein was the biological activity of the native protein towards the affinity resin and not physical differences between the native and denatured proteins.
Lebing and Chen, co-owned U.S. Pat. No. 5,610,285, described a method where xcex1-1 PI was captured from IV-1 paste suspension using a DEAE chromatography step. The collected solution was ultrafiltered then applied to an S-cation column for initial purification. Alpha-1 PI was collected as the flow-through fraction. The product, in a sucrose solution, was then treated with TNBP/cholate in order to inactivate viruses. Following filtration and ultrafiltration, the product was applied to a second S-cation column for final purification. Once the product was formulated and freeze dried, it was virally inactivated a second time by heating to 80xc2x0 C. for 72 hours. Product purity and virus safety were greatly improved versus the Hein process, described above, but, in practice, the Lebing and Chen process was too resource intensive for large-scale manufacturing.
A process for the purification of xcex1-1 PI that improves the yield and purity of the xcex1-1 PI, that requires a shorter production time and uses less resources (such as reagents, water, resins, and column size) is needed. The present invention provides a process of purifying xcex1-1 PI from a blood plasma fraction with a higher yield, higher purity, shorter production time, and use of less resources than known methods.
The present invention provides methods for purifying xcex1-1 PI from an aqueous solution containing xcex1-1 PI. A portion of contaminating proteins is removed from the aqueous solution, so that a partially purified solution containing xcex1-1 PI is obtained. The contaminating proteins may, for example, be precipitated by adding polyethylene glycol (PEG) to the aqueous solution and adjusting the pH of the solution to from about 5.0 to about 6.0. The conductivity of the partially purified solution is then adjusted, such as by diluting it to reduce its conductivity, for example. The purified solution is diluted with water, for example, and the water may contain sodium phosphate. The conductivity of the solution is adjusted so that xcex1-1 PI will bind to an anion exchange resin. This conductivity is typically between about 2.0 milliSiemens (mS) and about 6.0 mS, for example. The method further includes passing the purified solution over an anion exchange resin so that the xcex1-1 PI in the solution binds to the anion exchange resin. The xcex1-1 PI can then be eluted from the anion exchange resin to obtain an eluted solution containing xcex1-1 PI.
In another embodiment of the invention, a portion of contaminating proteins is removed by washing the anion exchange resin, to which xcex1-1 PI is bound, with a buffer solution. The buffer solution removes a portion of contaminating proteins that are bound to the anion exchange resin without removing xcex1-1 PI bound to the anion exchange resin.
In another embodiment of the invention, viruses are deactivated prior to dilution of the purified solution and its addition to an anion exchange resin. The viral inactivation occurs by, for example, adding a detergent to the purified solution to obtain a mixture of purified solution and detergent and adjusting the pH of the mixture to from about 6.5 to about 8.5. The detergent is preferably a non-ionic detergent, such as Tween 20, for example.
In a further embodiment, the method of the invention also includes a viral removal step. The viral removal step occurs by filtration of the diluted, purified solution, such as, for example, by nanofiltration.
In another embodiment, the method further includes passing the eluted solution obtained from the anion exchange resin through a cation exchange resin. The pH, conductivity, and protein concentration of the eluted solution are adjusted so that xcex1-1 PI will not bind to the cation exchange resin. The solution is then passed over the cation exchange resin, and a flowthrough that contains xcex1-1 PI is collected.
In other embodiments of the invention, xcex1-1 PI is purified from Fraction IV-1 of the Cohn-Oncley fractionation procedure. The Cohn Fraction IV-1 may be suspended in an aqueous solution for purposes of the purification process. An embodiment of the invention provides for removing a portion of contaminating proteins from a Cohn Fraction IV-1 to obtain a purified solution containing xcex1-1 PI, adjusting the conductivity of the purified solution so that xcex1-1 PI will not bind to an anion exchange resin, passing the purified solution through a n anion exchange resin, and eluting xcex1-1 PI from the anion exchange resin to obtain an eluted solution containing xcex1-1 PI.
Methods of the present invention provide for the isolation of xcex1-1 PI from aqueous solutions, such as human plasma, at yield and purity levels far above known processes. The methods of the present invention provide for a yield that is 50% greater than yields obtained with current production procedures and provides xcex1-1 PI at a purity that is increased to greater than 95%. The methods of the invention can produce a yield of xcex1-1 PI of at least 75% from Cohn Fraction IV-1 starting material. Methods of the invention can produce xcex1-1 PI having a purity of at least 98% by imimunonephelometry. Finally, methods of the invention also reduce the production time for the purification of xcex1-1 PI by approximately 40 hours to a production time no longer than 40 hours. These and other aspects of the invention will be made more apparent from the following description and claims.
The present invention provides a process for purifying xcex1-1 PI from an aqueous solution containing xcex1-1 PI, such as human plasma, for example. In methods of the invention, contaminating proteins are removed from the aqueous solution before it is passed through an anion exchange resin. Prior to passing the solution through the anion exchange resin, the conductivity of the solution is adjusted, preferably by diluting the solution, so that the xcex1-1 PI will bind to the anion exchange resin. This typically occurs at a conductivity of between about 2.0 mS and about 6.0 mS. The xcex1-1 PI is then selectively eluted from the anion exchange resin to provide xcex1-1 PI at yield and purity levels above those obtained with current processes.
A known procedure for the purification of xcex1-1 PI begins with Fraction IV-1 paste, as obtained through the Cohn-Oncley fractionation procedure for human plasma. See, e.g., E. J. Cohn, et al., J. Amer. Chem. Soc., 68, 459 (1946); E. J. Cohn, U.S. Pat. No. 2,390,074; and Oncley, et al., J. Amer. Chem. Soc., 71, 541 (1949) the entire disclosures of which are hereby incorporated by reference herein. The Cohn-Oncley process involves a series of cold ethanol precipitation steps during which specific proteins are separated according to isoelectric point by adjusting pH, ionic strength, protein concentration, temperature and ethanol concentration. The Fraction IV-1 paste obtained by this procedure is dissolved in a buffer solution and heated to activate xcex1-1 PI. An initial purification step includes the precipitation of contaminating proteins and lipids from the dissolved Fraction IV-1. The xcex1-1 PI is then precipitated from the dissolved Fraction IV-1 solution, and the crude xcex1-1 PI is passed through an anion exchange resin to remove contaminating proteins. A viral inactivation is accomplished by pasteurization for 10 hours at 60xc2x0 C. in a sucrose solution. Following pasteurization, the xcex1-1 PI is diafiltered, bulked in NaCl/Na3PO4, sterile filtered, and lyophilized.
In a preferred embodiment of the invention, the starting material for the process is Cohn Fraction IV-1, although other fractions, such as Cohn Fraction II+III, for example, can be used as a starting material. This fraction may be dissolved in an aqueous solution, such as a tris-(hydroxymethyl)amino methane (Tris) buffer solution, for example. Fraction IV-1 is typically a paste that can be dissolved in Tris buffer at a pH of about 9.25 to about 9.5 at about 40xc2x0 C. A salt, such as sodium chloride (NaCl) may also be added to the solution.
The process of the invention includes the removal of at least a portion of contaminating proteins from the aqueous solution to obtain a purified solution that contains xcex1-1 PI. Such contaminating proteins may include fibrinogen and albumin, for example. The portion of contaminating proteins is preferably removed by precipitation with a polyalkylene glycol, such as polyethylene glycol (PEG) or polypropylene glycol (PPG), for example. Other alcohols that are known to those of skill in the art to have similar properties may be used. PEG, the preferred polyalkylene glycol for use in methods of the invention, has a molecular weight of between about 2,000 and about 10,000, and preferably has a molecular weight of between about 3,000 and about 4,000. The PEG added to the solution is at least about 2% weight per volume of the mixture formed, is preferably about 3% to 15%, and is most preferably 11.5%. The pH of the solution may also be adjusted to precipitate the contaminating proteins. The pH is typically adjusted to between about 5.0 and about 6.0. The pH of the solution is adjusted by addition of an acid, such as acetic acid. The precipitate may then be separated from the solution by filtration, centrifugation, or any other conventional means known in the art, to obtain a filtrate containing xcex1-1 PI.
The conductivity of the filtrate is then adjusted prior to passing the filtrate over an anion exchange resin. The equilibrium between an ion exchange resin and a protein solution is influenced by the ionic strength of the solution (see, e.g., Yamamato, et al., Biotechnol. Bioeng., 25:1373-91 (1983)). The conductivity of the filtrate is therefore adjusted so that the xcex1-1 PI in the filtrate will bind to an anion exchange resin. This conductivity is typically between about 2.0 mS and 6.0 mS when measured at 25xc2x0 C., but other ranges of conductivity may be necessary to bind the xcex1-1 PI to an anion exchange resin. The conductivity is preferably adjusted by dilution of the filtrate, and not by gel filtration, diafiltration, or other means of salt removal. The filtrate is preferably diluted with water, which may contain sodium phosphate (Na3PO4), or other buffers capable of providing a pH of about 6-7.
After dilution of the filtrate, the solution is applied directly to an anion exchange resin. Unlike known methods, as described above, the filtrate is not subjected to further PEG precipitation or diafiltration prior to chromatographic separation. The diluted filtrate is passed over an anion exchange resin, which is preferably a quaternary aminoethyl (QAE) resin. While QAE chromatography is preferred, other anion exchange resins, such as trimethylamino ethane (TMAE) and diethyl aminoethyl (DEAE), may be used in methods of the invention. The xcex1-1 PI binds to the anion exchange resin. In a preferred embodiment, the anion exchange resin is washed with a buffer solution, such as an Na3PO4 buffer, to remove another portion of contaminating proteins. The proteins typically removed are albumin and transferrin. During the buffer wash, xcex1-1 PI remains bound to the anion exchange resin. After the buffer wash, xcex1-1 PI is eluted from the anion exchange resin to obtain an eluted solution containing purified xcex1-1 PI. Ceruloplasmin remains bound to the column during both the wash and elution.
A further purification of the protein may be accomplished by passing the eluted solution containing the xcex1-1 PI through a cation exchange resin. The pH, conductivity, and protein concentration of the eluted solution are adjusted so that xcex1-1 PI does not bind to the cation exchange resin. The influences of pH, conductivity, and protein concentration on the binding of xcex1-1 PI to a cation exchange resin are set forth in co-owned U.S. Pat. No. 5,610,825, the entire disclosure of which is hereby incorporated by reference herein.
Viral inactivation and/or viral removal also play a part in the purification of xcex1-1 PI from aqueous solutions, such as human plasma, for example. Known processes for the purification of xcex1-1 PI utilize a dry heat treatment for the inactivation of viruses. This treatment can denature xcex1-1 PI protein, however, thereby reducing the yield and/or purity of the xcex1-1 PI. The methods of the invention deactivate and remove viruses without this heat treatment step, thereby increasing both yield and purity of xcex1-1 PI obtained.
The above-described precipitation of contaminating proteins with 11.5% PEG also serves as one of the virus removal steps. Precipitation with 11.5% PEG removes both enveloped and non-enveloped viruses from the blood plasma fraction. This precipitation removes, with a xe2x89xa74 logs of clearance, at least four viruses, including HIV-1, BVDV, PRV, and Reovirus Type 3. In comparison, the dry heat process of known methods only results in xe2x89xa74 logs of clearance of three of these viruses; the Reovirus Type 3 is only removed at 1 log clearance. Additionally, the 11.5% PEG precipitation step has been shown to result in xe2x89xa74 logs of clearance of transmissible spongiform encephalopathies (i.e., TSE prions) from the blood plasma fraction. (See co-owned, co-pending application entitled Method of Separating Prions from Biological Material, filed on even-date herewith.)
Another viral deactivation is accomplished by addition of a non-ionic detergent to the aqueous solution. This step is preferably taken prior to passing the solution through the anion exchange resin. Non-ionic detergents for use in methods of the invention include, but are not limited to, Tweens, such as Tween 20 and Tween 80. Tween 20 is the preferred non-ionic detergent for use in methods of the invention. Tween 20 may be added at from about 0.33% to about 10% weight per volume of resulting mixture. Tween 20 is preferably added in the range of about 0.5% to about 2.0% and is most preferably added at 1.0%. The detergent treatment with 1% Tween 20 reduces enveloped viruses by  greater than 4 logs of clearance.
Another embodiment of the invention includes virus removal. Both enveloped and non-enveloped viruses are removed by filtration, preferably by nanofiltration, or any other filtration methods known in the art. In a preferred embodiment, the solution eluted from the ion exchange resin, which includes xcex1-1 PI, is subjected to nanofiltration. Nanofiltration reduces both enveloped and non-enveloped viruses by  greater than 4 logs of clearance.
The methods of the current invention, therefore, preferably include two  greater than 4 logs of clearance steps for the removal of enveloped viruses and non-enveloped viruses. These viral clearance levels are greater than those obtained by known methods of manufacture of xcex1-1 PI, which only include two virus clearance steps.
Practice of the invention will be understood more fully from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.